Methods using genetic package display for selecting internalizing ligands for gene delivery

ABSTRACT

A genetic package display system is presented for selecting internalizing ligands for gene delivery. The genetic package carries a reporter, selectable marker, or a specifically detectable nucleic acid sequence and presents a ligand on its surface. More specifically, a library of potential ligands may be screened for the ability to successfully transduce target cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 09/193,445,filed Nov. 17, 1998, and U.S. Ser. No. 09/195,379, filed Nov. 17, 1998,both pending, and which are continuation-in-part applications of U.S.Ser. No. 09/141,631, filed Aug. 28, 1998, now pending; which applicationclaims the benefit of priority from U.S. Provisional Application SerialNo. 60/057,067, filed Aug. 29, 1997, now abandoned.

TECHNICAL FIELD

This invention relates generally to genetic package display (e.g., phagedisplay), and in particular, to selection of ligands that bind to a cellsurface receptor and internalize.

BACKGROUND OF THE INVENTION

Bacteriophage expressing a peptide on its surface has been used toidentify protein binding domains, including antigenic determinants,antibodies that are specifically reactive, mutants with high affinitybinding, identify novel ligands, and substrate sites for enzymes. In itsmost common form, a peptide is expressed as a fusion protein with acapsid protein of a filamentous phage. This results in the display ofthe foreign protein on the surface of the phage particle. Libraries ofphages are generated that express a multitude of foreign proteins. Theselibraries are bound to a substrate or cell that presents the bindingpartner of interest. This screening process is essentially an affinitypurification. Bound phage are recovered, propagated, and the geneencoding the foreign protein may be isolated and characterized. Thistechnology is commonly referred to as “phage display.”

Through a process called “biopanning,” the specific phage carrying apeptide or protein that interacts with a protein or other moiety on asolid phase can be identified and isolated. However, in manyapplications, binding or binding affinity is not the sole criticalparameter. For example, in gene therapy, a gene sequence needs to beintroduced into a cell. In preferred methods, the gene sequence istargeted to particular cells by way of a ligand I cell surface receptorinteraction. Thus, the ligand must not only bind to the cells but mustalso be internalized. A native ligand that is internalized, when used ina system for gene therapy may not be efficiently internalized. Forexample, both FGF2 and EGF are internalizing ligands.

Phage libraries can be screened for potentially internalizing ligands bybiopanning on live cells and rescuing internalized phage from the cellsafter stripping off externally bound phage (erg., acid elution).However, this method may result in recovery of undesired phage that bindvery tightly or are only partially internalized. Moreover, phage thatare internalized and subjected to proteases lose infectivity and can notbe recovered. Accordingly, current methodologies are inadequate todetermine the usefulness of ligands for gene therapy.

Further, identification of target cells or tissues that are able tointernalize ligands and express a transgene would readily allow one toidentify specific target cells for known or putative ligands as well asallow one to identify ligands for specific cell or tissue types.However, current methods of target cell identification are hampered bythe same difficulties, as noted above, with regard to screening forinternalizing ligands. Accordingly, current methodologies are inadequateto determine which cell or tissue types are useful targets for ligandmediated gene therapy.

Thus, current screening methods are inadequate for selecting peptide orprotein ligands that bind to a cell surface receptor and internalize.The present invention discloses a display methods that select peptide orprotein ligands that internalize, and further provides other relatedadvantages.

SUMMARY OF THE INVENTION

Within one aspect of the present invention, a method of selectinginternalizing ligands displayed on a genetic package is presented,comprising: (a) contacting a ligand displaying genetic package(s) with acell(s), wherein the package carries a gene encoding a detectableproduct which is expressed upon internalization of the package; and (b)detecting product expressed by the cell(s); thereby selectinginternalizing ligands displayed on a genetic package.

In another aspect, the invention provides a method of identifying aninternalizing ligand displayed on a genetic package, comprising: (a)contacting one or more ligand displaying genetic packages with acell(s), wherein each package carries a gene encoding a detectableproduct which is expressed upon internalization of the package, (b)detecting product expressed by the cell(s); and (c) recovering a nucleicacid molecule encoding an internalizing ligand from the cell(s)expressing the product, and thereby identifying an internalizing liganddisplayed on a genetic package.

In yet another aspect, the invention provides a method of identifying aninternalizing ligand displayed on a genetic package, comprising: (a)contacting one or more ligand displaying genetic packages with acell(s), wherein each package carries a gene encoding a selectableproduct which is expressed upon internalization of the package, (b)incubating the cell(s) under selective conditions; and (c) recovering anucleic acid molecule encoding an internalizing ligand from the cell(s)which grow under the selective conditions; thereby identifying aninternalizing ligand displayed on a genetic package.

In yet another aspect, a method is provided for a high throughput methodof identifying an internalizing ligand displayed on a genetic package,comprising: (a) contacting one or more ligand displaying geneticpackages with a cell(s) in an array, wherein each package carries a geneencoding at least one detectable product which is expressed uponinternalization of the package; and (b) detecting product(s) expressedby the cell(s) in the array, and thereby identifying an internalizingligand displayed on a genetic package. In one embodiment, the liganddisplaying package comprises a library of ligand displaying packages.

In another aspect, the present invention provides a method ofidentifying an internalizing ligand displayed on a genetic package,comprising: (a) contacting one or more ligand displaying a geneticpackages with a cell(s), wherein each package carries a selectablemarker which is detectable upon internalization of the package, (b)detecting the selectable marker internalized by the cells; and (c)recovering a nucleic acid molecule encoding an internalizing ligand fromthe cell(s) carrying the selectable marker, thereby identifying aninternalizing ligand displayed on a genetic package.

In related embodiments, the selectable marker is selected from reportergene expression, expression of a gene that confers the ability to permitcell growth under selection conditions, non-endogenous nucleic acidsequences that permit PCR amplification, and nucleic acid sequences thatcan be purified by protein/DNA binding.

In preferred embodiments, the ligand displaying genetic packagecomprises a bacteriophage. The bacteriophage are filamentous phage orlambdoid phage in other preferred embodiments. In some embodiments, thebacteriophage carries a genome vector. In other embodiments, thebacteriophage carries a hybrid vector.

In other embodiments, the library is a cDNA library, an antibody genelibrary, a random peptide gene library, or a mutein library. In otherpreferred embodiments, the detectable product is selected from the groupconsisting of green fluorescent protein, β-galactosidase, secretedalkaline phosphatase, chloramphenicol acetyltransferase, luciferase,human growth hormone and neomycin phosphotransferase.

In other embodiments, the cells may be isolated by flow cytometry, forexample. In further embodiments, the methods further comprise recoveringa nucleic acid molecule encoding the ligand from the cell(s) expressingthe product.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth below whichdescribe in more detail certain procedures or compositions (e.g.,plasmids, etc.), and are therefore incorporated herein by reference intheir entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematic representations of phage vectors formammalian cell transduction. FIG. 1A depicts the parent phage vectorwith wild type pIII coat protein. The base vector is M13 genome withampicillin resistance (AmP^(R)) gene and GFP expression cassetteinserted into the intergenic region between pIV and pII (MEGFP3). TheMEGFP3 vector contains the following elements: ori-CMV, SV40 replicationorigin and CMV promoter; EGFP, enhanced green fluorescent protein gene;BGH, and a bovine growth hormone polyadenylation sequence. FIG. 1Brepresents the FGF-pIII fusion display phage (MF2/1G3).

FIG. 2 is a scanned image of a Western Blot analysis representingdetection of FGF2-pIII fusion protein in protein extracts from purifiedFGF2-phage (FGF2-MEGFP).

FIGS. 3A and 3B are bar graphs of ELISA detection of FGF2 on FGF2-phage.FIG. 3A depicts the amount of phage protein detected using both theempty MEGFP3 vector and the FGF2 fusion construct (FGF2-MEGFP). FIG. 3Bdepicts the amount of FGF2 detected on the phage having the fusionconstruct.

FIGS. 4A and 4B are bar graphs representing the transduction of COScells by FGF2-phage.

FIG. 5 is a bar graph representing the transduction of COS cells bypeptide display phage.

FIG. 6 is a scanned image of a Western Blot analysis representingdetection of EGF-pIII fusion protein in protein extracts from purifiedEGF-phage.

FIG. 7 is a bar graph representing the dose response of COS cells tovarious phage titers.

FIG. 8 is a bar graph representing a time course analysis of variousincubation times and the effect on transduction.

FIGS. 9A and 9B are bar graphs representing the specificity oftransduction of COS cells by EGF-phage.

FIG. 10 is a bar graph representing transduction specificity of avariety of human carcinoma cells.

FIG. 11 is a scanned image of ethidium bromide stained gelelectrophoretic analysis of products obtained by PCR amplification ofpIII genes/pIII gene fusions following various rounds of selection.

FIG. 12 is a vector map of an AAV-Phage hybrid genome vector.

FIG. 13 is a vector map of an AAV-Phage hybrid phagemid vector.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides methods of using liganddisplaying genetic packages to identify ligands that bind andinternalize. While it should be understood that a variety of liganddisplay methods may be utilized (e.g., phage display, RNA-peptidefusions, and ligand displaying bacteria), the present invention usesbacteriophage ligand display to exemplify the various embodiments.

Briefly, in one embodiment of the present invention, a library ofantibodies, cDNAs, or genes encoding random peptides is cloned into acoat protein (e.g., gene III protein of filamentous phage) of abacteriophage. The phage genome also contains an “expression cassette”encoding a transgene placed downstream from a cell promoter that isactive in the cells to be infected (FIG. 1A). The transgene is generallya selectable gene product and/or a detectable marker. Phage arecontacted with test cells and expression of the transgene is monitoredor selected. Phage that internalize will confer the phenotype of thetransgene, such as drug resistance or expression of a fluorescingprotein. The cells may be isolated on the basis of transgene expression.For example, when the transgene is a drug resistance gene, cells aregrown in the presence of the drug, such that only those cells receivingand expressing the transgene are propagated. The gene(s) that are fusedwith the coat protein and that promoted cell binding and internalizationare recovered from the selected cells by a suitable method.

I. DISPLAY PACKAGES

A variety of displaying genetic packages may be used within the contextof the present invention. A “ligand displaying genetic package” as usedherein, refers to any package which comprises a peptide/protein ligandand carries a nucleic acid molecule capable of detection, onceinternalized in the target cell. In one embodiment, the nucleic acidcarried by the ligand displaying genetic package is expressed uponinternalization into the cell thereby allowing for detection of aninternalized genetic package. In other embodiments, the liganddisplaying genetic package may carry a nucleic acid molecule whichallows for detection via PCR of unique sequences or by the ability ofthe internalized nucleic acid sequences to bind non-endogenous DNAbinding proteins (e.g., nucleic acid sequence could comprise a lacoperon, thereby allowing for lac repressor binding). Accordingly,display may be by a virus, RNA-peptide fusions, bacteriophage, bacteria,or similar system (See, Kay, Phage Display of Peptides and Proteins,pages 151-193, Academic Press, 1996). Preferred methods utilizebacteriophage. Such phage include the filamentous phages, lambda, T4,MS2, and the like. A preferred phage is a filamentous phage, such as M13or f1. Accordingly, many illustrations, while exemplifying the use ofphage, could also be performed with any ligand displaying geneticpackage.

Phage that present the foreign protein or peptide as a fusion with aphage coat protein are engineered to contain the appropriate codingregions. A variety of bacteriophage and coat proteins may be used.Examples include, without limitation, M13 gene III, gene VIII; fd minorcoat protein pIII (Saggio et al., Gene 152: 35, 1995); lambda D protein(Sternberg and Hoess, Proc. Natl. Acad. Sci. USA 92: 1609, 1995; Mikawaet al., J. Mol. Biol. 262: 21, 1996); lambda phage tail protein pV(Maruyama et al., Proc. Natl. Acad. Sci. USA 91: 8273, 1994; U.S. Pat.No. 5,627,024); fr coat protein (WO 96/11947; DD 292928; DD 286817; DD300652); φ29 tail protein gp9 (Lee, Virol. 69: 5018, 1995); MS2 coatprotein; T4 small outer capsid protein (Ren et al., Protein Sci. 5:1833, 1996), T4 nonessential capsid scaffold protein IPIII (Hong andBlack, Virology 194:481, 1993), or T4 lengthened fibritin protein gene(Efimov, Virus Genes 10:173, 1995); PRD-1 gene III; Qβ3 capsid protein(as long as dimerization is not interfered with); and P22 tailspikeprotein (Carbonell and Villaverde, Gene 176:225, 1996). Techniques forinserting foreign coding sequence into a phage gene are well known (seee.g., Sambrook et al., Molecular Cloning: A Laboratory Approach, ColdSpring Harbor Press, NY, 1989; Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Co., NY, 1995).

In the preferred filamentous phage system, a wide range of vectors areavailable (see, Kay et al., Phage Display of Peptides and Proteins: ALaboratory Manual, Academic Press, San Diego, 1996). The most commonvectors accept inserts in gene III or gene VIII. Furthermore, theforeign gene can be inserted directly into the phage genome or into aphagemid vector. Methods of propagation of filamentous phage andphagemids are well known.

Filarnentous phage vectors generally fall into two categories: phagegenome and phagemids. Either type of vector may be used within thecontext of the present invention. Many such commercial vectors areavailable. For example, the pEGFP vector series (Clonetech; Palo Alto,Calif.), M13mp vectors (Pharmacia Biotech, Sweden), pCANTAB 5E(Pharmacia Biotech), pBluescript series (Stratagene Cloning Systems, LaJolla, Calif.) and others may be used. One particularly usefulcommercial phagemid vector is pEGFP-N1, which contains a greenfluorescent protein (GFP) gene under control of the CMV immediate-earlypromoter. This plasmid also includes an SV40 origin of replication toenhance gene expression by allowing replication of the phagemid to highcopy number in cells that make SV40 T antigen.

Other vectors are available in the scientific community (see e.g.,Smith, in Vectors: A Survey of Molecular Cloning Vectors and their Uses,Rodriquez and Denhardt, eds., Butterworth, Boston, pp 61-84, 1988) ormay be constructed using standard methods (Sambrook et al., MolecularBiology: A Laboratory Approach, Cold Spring Harbor, N.Y., 1989; Ausubelet al., Current Protocols in Molecular Biology, Greene Publishing, NY,1995) guided by the principles discussed below.

The source of the ligand (e.g., gene, gene fragment or peptide encodingsequence) may be for example, derived from a cDNA library, antibodylibrary or random peptide library. Alternatively, the ligand may be froma library of random or selective mutations of a known ligand. In anadditional alternative, the ligand may be from a library of knownreceptor binding agents. For example, the library may contain a subsetof peptides known to bind the FGF or EGF receptor, but that have unknowngene delivery and expression characteristics (i.e. transductioncapacity).

When a cDNA library is used, the starting cDNA is synthesized from mRNAisolated from the source tissue or cell line from which the desiredligand originates. cDNA is then amplified using primers containingsequences of appropriate restriction enzyme sites for insertion into thedesired vector. Alternatively, commercially available cDNA libraries(e.g., Clonetech; Palo Alto, Calif.) may be amplified for insertion intothe vector.

Similarly, libraries of antibody fragments can be made from mRNAisolated from the spleen cells of immunized animals (immunized forexample with whole target cells or membranes) or subcloned from existingantibody libraries from immunized or naive animals. Random peptides aresubcloned from libraries that are commercially available (New EnglandBiolabs; Mass.) or can be synthesized and cloned using previouslydescribed methods (see, Kay et al., supra).

Phage display libraries of random or selective mutations of knownligands for improved gene delivery are performed in the same manner asdescribed for screening random peptide libraries such libraries arereferred to herein as a “mutein library” (i.e., a library of selectiveor random mutations). Random mutations of the native ligand gene may begenerated using DNA shuffling as described by Stemmer (Stemmer, Nature370: 389-391, 1994). Briefly, in this method, the ligand is amplifiedand randomly digested with DNase I. The 50-300 base pair fragments arereassembled in an amplification performed without primers and using TaqDNA polymerase or similar enzyme. The high error rate of this polymeraseintroduces random mutations in the fragments that are reassembled atrandom thus introducing combinatorial variations of different mutationsdistributed over the length of the gene. Error prone amplification mayalternatively be used to introduce random mutations (Bartell andSzostak, Science, 261:1411, 1993). The ligand may be mutated by cassettemutagenesis (Hutchison et al., in Methods in Enzymology 202:356-390,1991), in which random mutations are introduced using syntheticoligonucleotides and cloned into the ligand to create a library ofligands with altered binding specificities. Additional mutation methodscan be used. Some additional methods are described in Kay et al., supra.Further, selective mutations at predetermined sites may be performedusing standard molecular biological techniques (Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989).

If a cDNA library cannot be generated because, for example, the sourceof the desired ligand is not available or is unknown, random peptidelibraries or a cDNA library from placenta may be used as a startingpoint for screening. Methods for construction of random peptidelibraries may be found, for example, in Kay et al, supra. Briefly, therandom peptides are encoded by DNA assembled from degenerateoligonucleotides and inserted into one of the bacteriophage vectorsdescribed herein. Several different strategies may be used to generaterandom peptides. For example, triplets of NNN, wherein each N is anequimolar representation of all four nucleotides, will generate all 20amino acids (as well as 3 stop codons). Alternative strategies useNN(G/T) and NN(G/C), which results in 32 codons that encodes all 20amino acids and only 1 stop codon. Other strategies utilize synthesis ofmixtures of trinucleotide codons representing all 20 amino acids and nostop codons. Once the oligonucleotides are synthesized, they areassembled as double strands by a variety of schemes, one of whichinvolves synthesis of the complementary strand (see Kay et al., supra).

In addition to the ligand/coat protein fusion, in one embodiment, thevector may contain a gene whose product can be detected or selected for.As referred to herein, a “reporter” gene is one whose product can bedetected, such as by fluorescence, enzyme activity on a chromogenic orfluorescent substrate, and the like or selected for by growthconditions. Such reporter genes include, without limitation, greenfluorescent protein (GFP), β-galactosidase, chloramphenicolacetyltransferase (CAT), luciferase, neomycin phosphotransferase,secreted alkaline phosphatase (SEAP), and human growth hormone (HGH).Selectable markers include drug resistances, such as neomycin (G418),hygromycin, and the like.

The marker gene is in operative linkage with a promoter. Any promoterthat is active in the cells to be transfected can be used. The vectorshould also have a viral origin of replication and a packaging signalfor assembling the vector DNA with the capsid proteins.

Most applications of the present invention will involve transfection ofmammalian cells, including human, canine, feline, equine, and the like.The choice of the promoter will depend in part upon the targeted celltype. Promoters that are suitable within the context of the presentinvention include, without limitation, constitutive, inducible, tissuespecific, cell type specific, temporal specific, or event-specific,although constitutive promoters are preferred.

Examples of constitutive or nonspecific promoters include the SV40 earlypromoter (U.S. Pat. No. 5,118,627), the SV40 late promoter (U.S. Pat.No. 5,118,627), CMV early gene promoter (U.S. Pat. No. 5,168,062),bovine papilloma virus promoter, and adenovirus promoter. In addition toviral promoters, cellular promoters are also amenable within the contextof this invention. In particular, cellular promoters for the so-calledhousekeeping genes are useful (e.g., β-actin). Viral promoters aregenerally stronger promoters than cellular promoters.

In preferred embodiments, the phage has an origin of replicationsuitable for the transfected cells. Viral replication systems, such asEBV ori and EBNA gene, SV40 ori and T antigen, or BPV ori, may be used.Other mammalian replication systems may be interchanged. As well, thereplication genes may cause high copy number. Expression of therapeuticgenes from the phage genome may be enhanced by increasing the copynumber of the phage genome. In one method, the SV40 origin ofreplication is used in the presence of SV40 T antigen to cause severalhundred thousand copy number. The T antigen gene may be already presentin the cells, introduced separately, or included in the phage genomeunder the transcriptional control of a suitable cell promoter. Otherviral replication systems for increasing copy number can also be used,such as EBV origin and EBNA.

In one embodiment, a viral replication system such as an SV40 basedshuttle vector can be used as the phagemid. Accordingly, cells infectedby the ligand-expressing phage package the phagemid DNA into SV40 viralparticles. These viral particles infect neighboring cells, thusspreading the phagemid DNA. Following growth in culture, a dish of cellscontains millions of copies of the original phagemid, thereby enrichingthe population of internalized ligand encoding genetic packages severalfold. The cell lines used with such a system can either be transfectedwith DNA sequences encoding SV40 small and large T-antigens or cancontain the proteins through delivery with fusion constructs, such asVP22. VP22 is a herpes virus structural protein that is exported fromcells and spreads to neighboring cells where it concentrates in thenucleus (Elliot and O'Hare, Cell 88:223-233, 1997). VPP32-SV40 T antigenfusion protein encoding vectors exist in the art and are available fromInvitrogen Corp., as the pVP22myc-His vector. The vector useful in thisapplication ideally contains an f1, M13 or comparable phage origin, acoat protein fusion cassette (pIII or pVIII), the SV40 late regiongenes, and an SV40 origin. While the addition of a bacterial selectiongene would facilitate propagation in bacteria, it is not essential sincepropagation in large T containing cells should perform the necessaryamplification and thus selection.

Similarly, an adeno-associated virus/phage hybrid vector may be used toachieve the same amplified ligand production. An AAV-phage hybrid vectorcombines selected elements of both vector systems, providing a vectorthat is simple to produce in bacteria with no packaging limit, whileallowing infection of quiescent cells combined with integration into thehost chromosome. Vectors containing many of the appropriate elements arereadily available, and can be further modified by standard methodologiesto include the necessary sequences. For example, the phagemidpAAV/Svneo, ATCC Accession No. 68065, contains AAV ITR sequences and anF1 origin of replication. In addition the vector pAV.CMV.LacZ providesmany of the appropriate elements Fisher et al., J. Virol.70(1):520-532,1996.

Adeno-associated virus (AAV) is a defective member of the parvovirusfamily. The AAV genome is encapsulated as a single-stranded DNA moleculeof plus or minus polarity (Bems and Rose, J. Virol. 5:693-699, 1970;Blacklow et al., J. Exp. Med. 115:755-763, 1967). Strands of bothpolarities are packaged, but in separate virus particles (Bems andAdler, Virology 9:394-396, 1972) and both strands are infectious(Samulski et al., J. Virol. 61:3096-3101, 1987). The single-stranded DNAgenome of the human adeno-associated virus type 2 (AAV2) is 4681 basepairs in length and is flanked by inverted terminal repeated sequencesof 145 base pairs each (Lusby et al., J. Virol. 41:518-526, 1982;Muzyczka, Curr. Top. Microbiol. Immunol. 158:97-129, 1992). In addition,the viral rep protein appears to mediate non-homologous recombinationthrough the ITRs (Giraud et al., J. Virol. 69:6917-6924, 1995; Linden etal., Proc. Natl. Acad. Sci. USA 93:7966-7972, 1996). Accordingly, asparvoviral genomes have ITR sequences at each end which play a role inrecombination and which are generally required for parvoviralreplication and packaging, the vectors of the present inventiongenerally contain all or a portion of at least one of the ITRs or afunctional equivalent thereof.

Adeno-associated viruses may be readily obtained and their use asvectors for gene delivery has been described in, for example, Muzyczka,Curr. Top. Microbiol. Immunol. 158:97-129, 1992; U.S. Pat. No.4,797,368, and PCT Application WO 91/18088. Construction of AAV vectorsis described in a number of publications, including U.S. Pat. No.5,173,414; Lebkowski et al., Mol. Cell. Biol. 8:3988-3996, 1988;Tratschin et al., Mol. Cell. Biol. 5(11):3251-3260; Hermonat andMuzyczka, Proc. Nat'l. Acad. Sci USA 81:6466-6470, 1984; U.S. Pat. Nos.5,871,982, 5,773,289, 5,843,742, and 5,474,935; and PCT Application Nos.WO 98/45462 and WO 98/48005, all of which are incorporated herein byreference.

AAV-2 can be propagated as a lytic virus or maintained as a provirus,integrated into host cell DNA (Cukor et al., in “The Parvoviruses,”Berns ed., Plenum Publishing Corp., N.Y. pp. 33-66, 1984). Althoughunder certain conditions AAV can replicate in the absence of helpervirus (Yakobson et al., J. Virol. 61:972-981, 1987), efficientreplication requires coinfection with either adenovirus (Atchinson etal., Science 194:754-756, 1965; Hoggan, Fed. Proc. Am. Soc. Exp. Biol.24:248, 1965; Parks et al., J. Virol. 1:171-180, 1967); herpes simplexvirus (Buller et al., J. Virol. 40:241-247, 1981) or cytomegalovirus,Epstein-Barr virus, or vaccinia virus. Hence the classification of AAVas a “defective” virus.

The AAV-phage hybrid vector for ligand identification generallycomprises an F1, M13 or comparable origin, a coat protein fusioncassette (e.g., pIII-ligand or pVIII-ligand), a bacterial gene whichfacilitates selection, such as ampicillin resistance, and the two AAVITRs (or functional equivalents thereof), between which is inserted thepromoter driven reporter or selectable gene. The hybrid phage can thenbe used to transduce cells. The positive cells are identified and theligand sequences are amplified by PCR using the ITRs or coat proteingene as the template sequence. This amplified ligand fusion constructcan then be sub-cloned into other vectors for further rounds oftransfection, selection, and ligand sequence identification.

In related embodiments, the present invention provides an AAV-phagevector that is designed to produce functional AAV viral particles uponco-infection with a “rescue” virus, such as an adenovirus. In thisembodiment, the AAV-phage particle enters the cell as a “phageparticle”, but once inside, produces AAV particles that infectsurrounding cells. This method can thus be used to amplify gene deliveryeffectiveness in a target organ or tissue. Briefly, in this approach, aligand displaying phage particle containing a AAV-phage vector whichcontains a transgene of interest as well as ligand fused to the pIII orpVIII coat protein is used to transduce a cell. Concurrent with orsubsequent to contacting the cells with the transgene containingAAV-phage particle, a ligand displaying helper AAV-phage is also used tosupply the rep and cap genes for viral particle formation. Followingtransduction with the appropriate bacteriophage, the cell is infectedwith a “rescue” virus, such as an adenovirus, which allows viralparticles to form and infect neighboring cells. Alternatively, the repand cap functions can be supplied on the helper adenoviral genome.

In a related embodiment, mutant coat proteins may be used increasetransduction efficiency. A particularly preferred manipulation is tomutagenize a coat protein so as to facilitate uncoating upon cellularinternalization. For example, the filamentous phage coat protein VIIIencoding gene can be mutagenized such that it is encodes a slightlyunstable protein and thus allows more rapid uncoating and increasedtransduction capacity.

In other embodiments, peptides or other moieties that allow or promotethe escape of the vectors (and any molecule attached thereto or enclosedtherein) from the endosome are incorporated and expressed on the surfaceof the bacteriophage. Such “other moieties” include molecules that arenot themselves peptides but which have the ability to disrupt theendosomal membrane, thereby facilitating the escape of the vector, andmolecules that otherwise mimic the endosomal escape properties of thewithin-described peptide sequences (see, e.g., published PCT ApplicationNo. WO 96/10038, the disclosure of which is incorporated by referenceherein).

Peptide sequences that confer the ability to escape the endosome areparticularly preferred. Such sequences are well known and can be readilyfused covalently or genetically to a coat protein, such as gene III orgene VIII of filamentous phage. Although fusion of one or more peptidesequences to a coat protein is described herein as a preferredembodiment, it should be understood that other methods of attachment—andother moieties besides peptides—are useful as disclosed herein.

Thus, an example of a dual display filamentous phage presents a ligand(e.g., FGF) as a fusion to gene III and an endosomal escape peptidefused to gene VIII. The locations of the ligand and escape sequences areinterchangeable. Escape sequences that are suitable include, withoutlimitation, the following exemplary sequences: a peptide of Pseudomonasexotoxin (Donnelly, J. J., et al., PNAS 90:3530-3534, 1993); influenzapeptides such as the HA peptide and peptides derived therefrom, such aspeptide FPI3; Sendai Virus fusogenic peptide; the fusogenic sequencefrom IHV gp1 protein; Paradaxin fasogenic peptide; and Melittinfusogenic peptide (see WO 96/41606).

Another sequence that may be included in a vector is a sequence thatfacilitates trafficking proteins into the nucleus. These so-callednuclear translocation or nuclear localization sequences (NLS) aregenerally rich in positively charged amino acids. Because the carboxylterminus of gene VIII protein of filamentous phage already carries apositive charge, increased charge and likeliness of nuclear transportmay be enhanced by fusing known mammalian cell NLS sequences to the geneVIII protein. NLS fusions to other coat proteins of filamentous phagemay be substituted.

Examples of NLS sequences include those resembling the short basic NLSof the SV40 T antigen; the bipartite NLS of nucleoplasmin; theribonucleoprotein sequence A1; the small nuclear ribonucleoproteinsequence U1A, and human T-lymphocyte virus-1Tax protein. Other usefulNLS sequences include the HIV matrix protein NLS; and the nucleartranslocation components importain/hSRP1 and Ran/TC4; the consensussequence KXX(K/R) (SEQ ID NO.:4) flanked by Pro or Ala; the nucleartranslocation sequence of nucleoplasmin; or the NLS from antennapedia(see WO 96/41606).

Further, sequences which direct the genetic package to various cellularcompartments may be useful within the context of the present invention.For example, while FGF appears to be trafficked to the nucleus via anuclear localization-like peptide, EGF appears to be trafficked throughthe lysosome. Accordingly, in addition to the putative ligand, alysosomal directing sequence may be incorporated into one of the coatproteins of the genetic package. Exemplary sequences in this regard areKCPL which acts as a lysosomal targeting sequence (Blagoveshchenskaya etal., J. Biol. Chem. 273(43):27896-27903, 1998), the ubiquitin-dependentendocytosis motif DSWVEFIELD (Govers et al., EMBO J. 18(1):28-36, 1999,and DQRDLI or EQLPML from MCHII which also target the lysosome (Kang etal., J. Biol. Chem. 273(32):20644-20652, 1998).

As described herein, the library is then propagated in the display phageby transfection of a suitable bacteria host (e.g., DH5αF′ forfilamentous phages), and growing the culture, with the addition of areplication-competent helper virus if necessary, overnight at 37° C. Thephage particles are isolated from the culture medium using standardprotocols.

Infection of mammalian cells with phage is performed under conditionsthat block entry of wild type phage into cells (Barry et al., NatureMed. 2:299-305, 1996). Phage are added directly to cells, typically attiters of ≦10¹² CFU/ml in a buffer, such as PBS with 0.1% BSA or othersuitable blocking agents, and allowed to incubate with the cells at 37°C. or on ice. The amount of phage added to cells will depend in partupon the complexity of the library. For example, a phage display librarycontaining 10⁵ members has each member represented 10⁶ times in 1 ml ofa typical phage titer of 10¹¹ colony forming units/ml.

II. DETECTION/SELECTION OF TRANSGENE EXPRESSION

The genetic package display library is ultimately screened against thetarget tissue or cell line. Screening can be performed in vitro or invivo. While combinatorial screening methods have been performed in thepast, these methods are unable to determine the transduction capabilityof the displayed ligand (see, U.S. Pat. No. 5,733,731, incorporatedherein by reference). The criteria for a positive “hit” in the presentinvention is that the phage must be able to bind, be internalized, andenable detection of the internalized ligand by detecting a selectablemarker, such as, for example, by expressing the phage genomic DNAcontaining the reporter/selectable gene in the target or test cell orallowing direct nucleic acid detection (e.g., PCR or DNA binding). Inthis regard, it is believed that the phage should bind, internalize,uncoat, translocate to the nucleus, and replicate, in order to expressthe gene or otherwise facilitate detection (however, translocation anduncoating may occur in any order). Thus, in preferred embodiments onlyphage that reach the nucleus are selected.

The test cells may be any cells that express a receptor of choice or area cell type or source for which gene therapy is destined. Thus, in someinstances, the receptor may be unknown. In such cases, the selectionmethod can be used to isolate a ligand for a receptor without a knownligand (orphan receptor) such as erbB3. Briefly, the orphan receptor iscloned into a mammalian expression vector that also contains aselectable drug resistance gene and transfected into mammalian cells,such as COS cells. Stable transfectants that overproduce the orphanreceptor are selected by cultivation in the appropriate drug. Thisreceptor-transformed COS cell line is then used as the cell line forselection of ligand-displaying phage.

Tissue-specific or tumor-specific ligands can be selected bypre-absorption of the phage library against normal or non-targetedtissues of cell cultures. The selection process can also be applied invivo by injecting the library into tumor-bearing mice. The tumor isremoved from the mouse 48-72 h after injection. A cell suspension isprepared and phage genome bearing cells selected by one of the methodsdescribed herein. The gene whose product allows entry and expression ofthe phage genome is then isolated from the drug resistant cell colonies.

Screening may be performed directly against the target cells with nopre-screening or pre-enrichment. In one aspect, the present inventionprovides a method of identifying target cells or tissues for known orputative ligands. In this regard, phage display may be used to display alibrary of known or putative ligands (e.g., peptides, antibody fragmentsand the like) and screen singular tissues or cell types, or pools oftissues or cell types, thereby identifying target cells or tissues whichare effectively transduced by a ligand. As used herein, “pool” refers totwo or more cell types or tissue types. In one embodiment, known ligandsare presented on a ligand displaying genetic package to a pool of avariety of cell or tissue types and transgene expression is monitored.In a further embodiment, putative ligands are used to screen a pool of avariety of cell or tissue types for transduction ability. In this regardligands may be recovered and identified which efficiently transduce aparticular tissue or cell type. Identification of cell specific ligandscould greatly improve existing vectors for therapeutic gene delivery bytargeting specific cells thus reducing toxicity and allowing vectors tobe administered systemically.

Such cell type or tissue-type screening provides for selection thatrequires biological interaction rather than simple binding and does notrequire recovery of infective phage. In addition, cell surface receptorsneed not be identified and purified for the screening to be effective. Afurther aspect of the present invention is that it can be easily adaptedto high throughput applications for screening a variety of cell types ortissues and/or for screening libraries of putative ligands againstlibraries of putative receptors/binding partners (i.e., anti-ligands)which lead to transgene expression (see infra). In this regard,screening of a variety of ligand/cell interactions could be performed,including, for example, pathogen/host interactions, ligand/receptor,etc.

In one aspect, the present invention may be utilized to identify avariety of protein-protein interactions. In particular, a set of unknownproteins/peptides may be selected based upon interaction with anotherset of known or unknown proteins/peptides (e.g., random peptides, cDNAlibraries, or antibody gene libraries). In one embodiment, putativeligands are displayed on the surface of filamentous phage that carry areporter gene. These display phage are contacted with a cell linedisplaying a putative anti-ligand (protein/peptide) on its surface as areceptor fusion protein, such that binding of successful detection ofthe reporter gene requires binding of the phage display ligand and thecell surface displayed anti-ligand, as well as internalization andtransgene expression. Such screening can be utilized in a variety ofmethods, for example, a known ligand may be screened against a libraryof potential anti-ligands, a library of unknown ligands may be screenedagainst a known protein/peptide anti-ligand, and two libraries ofpeptides/proteins may be screened against each other to identifyligand/anti-ligand interactions (protein-protein).

A ligand/anti-ligand pair refers to a complementary/anti-complementaryset of molecules that demonstrate specific binding, generally ofrelatively high affinity. Exemplary ligand/anti-ligand pairs include anantibody and its ligand as well as ligand/receptor binding. While itshould be understood that the designation of either component of theabove mentioned ligand/anti-ligand pairs as either a ligand oranti-ligand is arbitrary, when necessary to specify a particularcomponent, a “ligand”, as used herein, is meant to describe peptides orproteins displayed on a genetic package carrying an expressibletransgene. Further, when necessary to define anti-ligand withspecificity, an “anti-ligand”, as used herein, demonstrates highaffinity and is expressed on the surface of the target cell to bemonitored for transgene expression.

Any cell surface receptor may be used as the fusion construct for thecell surface displayed anti-ligand. However, in a preferred embodiment,the extracellular domain of the receptor is replaced with the putativeanti-ligand. Construction of such fusions is routine in the art giventhat sequences as well as the extracellular intracellular domains ofnumerous receptors are known and available in the art. Komesli et al.,Eur. J. Biochem 254(3):505-513, 1998; Naranda et al., Proc. Natl. Acad.Sci. USA 94(21):11692-11697, 1997; Rutledge et al., J. Biol. Chem.266(31):21125-21130, 1991; Lemmon et al., Embo J. 16(2):281-294, 1997;Foehr et al., Immunol. Cell Biol 76(5):406-413, 1998. Exemplary fusionconstructs include, for example, anti-ligand-FGF receptor oranti-ligand-EGF receptor constructs.

In a further embodiment, a large pool of cDNAs may be tested bytransfecting into a large number of mammalian cells (e.g., COS cells).Ligand displaying phage are exposed to the transfected cells andpositive cells identified by either drug selection or detection of anexpressed transgene (e.g., GFP sorted by FACs). PCR may be performed onsingle cells to identify ligand/anti-ligand binding pairs. In thisregard PCR primers directed to the known portion of the fusion constructmay be used. For example, for phage display using pIII to display theligand, the PCR primer will be directed to the pIII gene, while in orderto identify the anti-ligand, the PCR primer will be directed to thesurface membrane protein (e.g., a receptor domain) encoding portion ofthe fusion construct. Alternatively, the plasmids within positive cellsmay be rescued by Hirt supernatant method and separated from phage DNAby gel electrophoresis or chromatography. (Kay et al., supra). Theselected cDNA plasmids may then be used to retransform bacteria. Newplasmid DNA is prepared and used for additional rounds of screening bytransfection into the cells and phage contact.

In an alternative embodiment, detection may be by any means which allowsfor the detection of the internalized nucleic acid molecules, and mayinclude Hirt extraction of small DNA, direct polymerase chain reaction(PCR) amplification of ligand DNA from reporter gene expressing cellsand non-endogenous protein-nucleic acid molecule binding interactions(e.g., lac operon and lac repressor in a mammalian cell) in positivecells. In addition, it is possible to use direct PCR amplification ofligand DNA from cells wherein no reporter gene is used.

In the various embodiments of the present invention utilizing PCRamplification of ligand sequences, the methodologies allow for the rapidamplification of only internalized sequences. Typical phage displaytechnologies require that the phage of interest (e.g., that which bindsto a particular target) be eluted and amplified following transductionof the appropriate host bacterial strain. However, transduction ofbacteria requires that bacteriophage are intact and maintaininfectivity. To eliminate the requirement for infective phage,methodologies provided herein allow those of ordinary skill in the artto recover, by PCR, phage DNA sequences that have been trafficked to thenucleus, digest these sequences with appropriate restriction enzymes,remove extraneous sequences, subclone the desired sequences back intothe phage display vector, and transform bacteria with this vector.Accordingly, by not requiring that the recovered phage be infective, theability to display larger ligands as fusion constructs with coatproteins is possible. Further, recovery of uncoated phage, such as thosetargeted to the lysosomal or endosomal compartments, as well as thosecapable of directing expression in the nucleus is possible.

Briefly, in one aspect, the recovery by PCR and amplification proceedsas follows: An initial selection of cells is performed using thedetection of a reporter gene, selective conditions, or the like and thetotal DNA is recovered from these cells. The recovered DNA is used as atemplate for PCR primers that are designed to flank the sequence ofinterest (the ligand encoding sequence, i.e., by using the pill or pVIIIsequences flanking the ligand insert as primer templates). The primerscan be manufactured such that they can be easily removed from thereaction mixture, for example, the primers may contain a biotin moietyat the 5′-end. In the alternative, the primers may be removed by anyknown methods, including, for example, gel extraction, selectiveprecipitation, and the like. In other alternatives primers need not beremoved, however their removal facilitates ligation efficiency. Further,the primers can provide restriction sites for subcloning etc.

Following amplification, the PCR product is purified to remove thepolymerase and digested with restriction enzyme to excise the putativeligand insert. Enzymes are chosen in order to facilitate directionalsubcloning into either the original vector or a new construct, if sodesired. Following enzyme digestion, the extraneous sequences areremoved (e.g., by using biotinylated primers and streptavidin conjugatedto beads or other solid support). The resulting DNA sequences are thenligated into the desired vector and the resulting vector is transformedinto bacteria using standard methodologies. The transformed bacteria arethen used to generate new phage particles or new DNA for additionalrounds of screening. However, while it should be understood that the useof a reporter gene may lead to enhanced DNA recovery and fewer rounds ofscreening, there is no requirement that a reporter gene be used.

In an alternative embodiment, recovery of replicated internalizednucleic acid molecules may be achieved via a nucleic acid bindingdomain. Accordingly, when using phage, the phage genome can be alteredsuch that a DNA binding sequence is incorporated therein. In oneexample, the phage vector may contain one or more copies of the lacoperon, thereby allowing any internalized and replicated phage vectorsto be purified from a cell lysate by a solid surface having conjugatedthereto the lac repressor protein. Briefly, target cells are contactedwith ligand displaying genetic packages (e.g., phage) for 48 to 72hours. Since only the packages displaying an appropriate ligand areinternalized and reach the cell nucleus where vector replication takesplace, these will be the sequences that will be selected for and thus,no reporter gene is required (e.g., GFP). Accordingly, the replicatedvector is double stranded and the double stranded form of the lac operonwill bind the lac repressor. The cells are then lysed and nuclearextracts are prepared, which are then passed over a solid support (e.g.,Sepharose 4B) having conjugated thereto the lac repressor protein. Thecolumn is washed, then eluted by a salt or pH gradient, therebyreleasing the bound DNA which can now be utilized in PCR reactions toamplify the ligand sequences for sub-cloning into another vector forfurther rounds of infection or characterization or the DNA can be useddirectly (without PCR) to transform bacteria and thereby produce morephage for further screening.

In other embodiments, the ligand displaying genetic package may alsocontain the nucleic acid sequences that encode the nucleic acid bindingprotein. For example, in the illustration above, the vector could alsoencode the lac repressor and the solid support has an anti-lac repressorantibody conjugated thereto, thereby allowing for recovery of nucleicacid molecules bound by the lac repressor.

One of ordinary skill in the art would readily recognize that a varietyof nucleic acid binding proteins could be utilized as described above.In this regard, many proteins have been identified that bind specificsequences of DNA. These proteins are responsible for genome replication,transcription and repair of damaged DNA. The transcription factorsregulate gene expression and are a diverse group of proteins. Thesefactors are especially well suited for purposes of the subject inventionbecause of their sequence-specific recognition. Host transcriptionfactors have been grouped into seven well-established classes based uponthe structural motif used for recognition. The major families includehelix-turn-helix (HTH) proteins, homeodomains, zinc finger proteins,steroid receptors, leucine zipper proteins, the helix-loop-helix (HLH)proteins, and P-sheets. Other classes or subclasses may eventually bedelineated as more factors are discovered and defined. Proteins fromthose classes or proteins that do not fit within one of these classesbut bind nucleic acid in a sequence-specific manner, such as SV40 Tantigen and p53 may also be used.

These families of transcription factors are generally well-known (seeGenBank; Pabo and Sauer, Ann. Rev. Biochem. 61:1053-1095, 1992; andreferences below). Many of these factors are cloned and the preciseDNA-binding region delineated in certain instances. When the sequence ofthe DNA-binding domain is known, a gene encoding it may be synthesizedif the region is short. Alternatively, the genes may be cloned from thehost genomic libraries or from cDNA libraries using oligonucleotides asprobes or from genomic DNA or cDNA by polymerase chain reaction methods.Such methods may be found in Sambrook et al., supra.

Helix-turn-helix proteins include the well studied λ Cro protein, λcI,and E. coli CAP proteins (see Steitz et al., Proc. Natl. Acad. Sci. USA79:3097-3100, 1982; Ohlendorf et al., J. Mol. Biol. 169:757-769, 1983).In addition, the lac repressor (Kaptein et al., J. Mol. Biol.182:179-182, 1985) and Trp repressor (Scheritz et al., Nature317:782-786, 1985) belong to this family. Members of the homeodomainfamily include the Drosophila protein Antennapaedia (Qian et al., Cell.59:573-580, 1989) and yeast MATα2 (Wolberger et al., Cell. 67:517-528,1991). Zinc finger proteins include TFIIIA (Miller et al., EMBO J.4:1609-1614, 1985), Sp-1, zif268, and many others (see generally Krizeket al., J. Am. Chem. Soc. 113:4518-4523, 1991). Steroid receptorproteins include receptors for steroid hormones, retinoids, vitamin D,thyroid hormones, as well as other compounds. Specific examples includeretinoic acid, knirps, progesterone, androgen, glucocosteroid andestrogen receptor proteins. The leucine zipper family was defined by aheptad repeat of leucines over a region of 30 to 40 residues. Specificmembers of this family include C/EBP, c-fos, c-jun, GCN4, sis-A, andCREB (see generally O'Shea et al., Science 254:539-544, 1991). Thehelix-loop-helix (HLH) family of proteins appears to have somesimilarities to the leucine zipper family. Well-known members of thisfamily include myoD (Weintraub et al., Science 251:761-766, 1991);c-myc; and AP-2 (Williams and Tijan, Science 251:1067-1071, 1991). Theβ-sheet family uses an antiparallel β-sheet for DNA binding, rather thanthe more common α-helix. The family contains the MetJ (Phillips, Curr.Opin. Struc. Biol. 1:89-98, 1991), Arc (Breg et al., Nature 346:586-589,1990) and Mnt repressors. In addition, other motifs are used for DNAbinding, such as the cysteine-rich motif in yeast GAL4 repressor, andthe GATA factor. Viruses also contain gene products that bind specificsequences. One of the most-studied such viral genes is the rev gene fromHIV. The rev gene product binds a sequence called RRE (rev responsiveelement) found in the env gene. Other proteins or peptides that bind DNAmay be discovered on the basis of sequence similarity to the knownclasses or functionally by selection. Furthermore, those of ordinaryskill in the art will appreciate that the nucleic acid binding domainwill chosen for a particular recovery method will preferably be onewhich is not already present within the target cells.

In a further embodiment, known or putative ligand-display phage may beused to screen a panel of cells that each express a potential targetreceptor. The source of the target receptor may be a known (i.e. cloned)receptor cDNA, or a collection of putative receptor cDNAs. For example,the putative receptor cDNAs may be identified from an epitope-taggedcDNA library as cDNAs that encode proteins that appear on the surface ofcells. (see, Sloan et al., Protein Expression and Purification11:119-124, 1997). Such cDNAs are inserted into an appropriate mammalianexpression vector and transfected into a host cell. Preferably the hostcell is eukaryotic, and more preferably the host cell is mammalian. Theexpression of the cDNA may be either stable or transient. Followingexpression the cells are contacted with the ligand-display phage andmonitored for transgene expression (e.g. drug resistance, GFP, or otherdetectable product). One skilled in the art would recognize thatidentification of cell or tissue types as described above, in additionto using ligand display phage, could also performed by utilizing otherligand displaying means, such as RNA-peptide fusions as described byRoberts and Szostak (Proc. Nat. Acad. Sci. USA 94:12297-12302, 1997),other phage types, or on bacteria.

Pre-screening or pre-enrichment may be used and can be especiallyhelpful when either too few or too many hits are observed. Enrichmentfor cell binding may improve detectability if no hits are found in theinitial screen. A prescreen to remove phage that bind non-specific cellssurface proteins may reduce non-specific hits if there are too manyinitial hits. For example, infection of 10⁷ target cells is performedwith about 10¹¹ phage, however a variety of cell density and phage titerranges are useful. The cells are incubated for at least 2 hours inPBS/BSA and washed extensively (Barry et al., Nature Med. 2:299-305,1996). The cells are incubated in media at 37° C. for 48-96 hours andthen detected or selected on the basis of expression of the reportergene.

Assays for each of these reporter gene products are well known. Forexample, GFP is detected by fluorescence microscopy or flow cytometry,SEAP is detected in medium using a fluorescent substrate (Clonetech;Palo Alto, Calif.), human growth hormone may be detected in medium by asimple and sensitive radioimmune assay (Nichols Institute; Calif.).Western blotting and ELISA may also be used to immunologically detectand measure the presence of reporter gene product. Alternatively, themessage for the reporter gene is detected using RNase probe protectionor fluorescent probe hybridization. For isolation of the phage vectorDNA and insert, any technique that can identify and isolate the cellsexpressing detectable marker product may be used. Flow cytometry, inparticular, is well suited for detecting fluorescence in or on a celland isolating that cell. Further, flow cytometry is well suited for highthroughput methodologies when necessary to isolate individual cells orgroups of cells that express a reporter gene.

When the reporter gene is a selectable marker, the cells are grown inselective conditions. Depending upon the marker, the conditions may be aparticular growth temperature, addition of a drug, or the like. In theexamples provided herein, the selectable marker is neomycin transferase,which confers G418 resistance on mammalian cells. Briefly, the cells aregrown in the presence of G418 for 7-14 days or until resistant coloniesare visible microscopically. Colonies are picked, and phage vector DNArecovered, conveniently as amplification of the insert.

Alternatively, multiple rounds of infection and selection are performedto reduce the complexity of the infecting phages. For example,drug-resistant colonies are pooled and the selected inserts amplifiedand cloned back into the phage display vector for a new round ofinfection. When the reporter is fluorescent, flow cytometry can be usedto select the strongest fluorescing cells to select the most highlyefficient gene delivery ligands. More stringent screening conditionsalso include higher selective drug concentrations. At the completion ofa selection process, representative phage clones may be subjected to DNAsequence analysis to further characterize gene delivery ligands.

In a further aspect, high throughput screening methodologies, such asscreening libraries by sub-selection of pools, may be utilized toidentify ligands. Briefly, phage stocks containing a variety of members,as individual plaques, may be used in combination with an array toidentify potential internalizing ligands. For example, a stock ofbacteriophages containing library members may be divided into subsetpool stocks such that each stock contains about 10² to about 10³members. Each stock solution is then screened utilizing an array (e.g.,multi-well plates containing target cells). Upon detection of a reportergene the phage stock may be sub-divided again and screened repeatedlyuntil the phage which contains the internalizing ligand is identified.Alternatively, those of skill in the art will appreciate that the arraymay contain a variety of cell types which are capable of being screenedwith one or more phage libraries, of which may also include a variety ofreporter genes (if so desired). For example, a variety of alternativelycolored fluorescent protein expression vectors are available which canbe used as reporter genes to provide multiplexing capability (Clonetech,Palo Alto, Calif.). Accordingly, rapid identification of those cellswhich internalize the bacteriophage and/or libraries that containinternalizing ligands for a specific cell type, may be identified.Utilizing both a variety of bacteriophage libraries as well as a varietyof cell types, would allow for a high throughput method of determiningsubsets of libraries that contain ligands for specific cell types,simultaneously. Array's for binding biomolecules are known in the artand therefore could be adapted to utilize the phage screeningmethodology of the present invention, see, e.g., PCT Application No. WO95/11755, PCT Application No. WO 95/35505, U.S. Pat. No. 4,591,570. Inaddition, affinity based biosensors such as a Biacore instrument,available commercially from Biacore AB, Uppsula, Sweden, may be used toimmobilize phage or cells for high throughput screening.

Moreover, while commonly used high throughput methodologies whichutilize live cells are typically performed on arrays of 6 to 96 wellplates, the current invention may also be carried out using cellularmicro-arrays such as those described by U.S. Pat. No. 5,776,748.Briefly, such arrays may be manufactured such that designated areas ofthe array bind a defined number of cells or size of tissue. For example,the arrays can be constructed such that they bind only a single cell.Therefore, an array of single cells may be constructed with a variety ofcell or tissue types. Because the size of the cell binding islands onthe array may be chosen such that no more than one cell may bind on anygiven island, because the locations and geometric pattern of the islandsmay be predetermined, and because the cells will remain at fixedlocations during assaying, cellular micro-arrays can provide for a highefficiency and high throughput method of assaying for internalizingligands, anti-ligands, or target cells or tissues.

In a preferred embodiment flow cytometry is utilized, the cells areidentified and counted by an automated detector unit. Because thelocations and geometric patterns of the islands are predetermined, thedetector can be designed or programmed to take measurements specificallyat those locations. Therefore, identification of individual cells whichhave been successfully transduced by a ligand displaying genetic packagecarrying a nucleic acid molecule which encodes a detectable product iseasily accomplished. In some embodiments, cells transduced by a liganddisplaying genetic package carrying a nucleic acid molecule whichencodes a selectable marker may be first selected on the basis of theappropriate sensitivity or resistance and then plated as individualcells and further selected or characterized by the methods describedherein. In particular, selection may be employed prior to plating on theplates to isolate transformed or transfected cells and then the cellsmay be assayed in situ.

In addition, when using fluorescence assays, a detector unit may beplaced above the plate or, if the plate is translucent, below the plate.In the case of transmission spectrophotometric assays, a translucentplate is used, a source of electromagnetic radiation is placed on oneside of the plate and a detector unit on the other. Because of the smalldistances between individual isolated cells permitted by the presentinvention, detectors employing fiber optics are particularly preferred.Such sources of electromagnetic radiation and such detectors forelectromagnetic transmission, reflection or emission are known in theapplicable art and are readily adaptable for use with the inventiondisclosed herein.

Screening in vivo may be performed similar to methods for targetingorgans or xenograft tumors using phage displayed peptides (Pasqualini etal., Nature Biotech. 15: 542-546, 1997; Pasqualini et al., Nature 380:364-366, 1996), except that the tissues, organs, or tumors are examinedfor reporter gene expression instead of the presence of phage. Briefly,a phage display library is injected intravenously into animals,generally mice, and organs or tumor samples are tested for reporter genefunction at 48-96 hours after injection. Tumor cells may be cultured inselective conditions or sorted by flow cytometry or other method toenrich for cells that express the phage transducing gene. The ligandencoding sequences can be amplified from selected cells as describedabove. As in in vitro screening, repeated rounds of infection andre-screening, alone or in combination with increased screeningstringency, may be used to obtain the most efficient gene deliveryligands.

Specificity may also be examined in vitro using a panel of non-targetedand targeted cell lines and detecting expression of the phagetransducing gene. Competition studies with free ligand or a neutralizingantibody to the ligand or receptor are used to confirm specific entry ofphage via the ligand receptor complex. Alternatively, the clonedreceptor for the ligand can be overexpressed in a cell line thatnormally does not express that receptor. Phage internalization andexpression into the stable transfectants expressing the receptor but notthe parent cell line indicates the specificity of the ligand for itsreceptor on receptor bearing cells.

Ligands that are identified as gene targeting ligands using theselection strategies described herein may be further tested forspecificity by reporter gene expression in target and non-target cellsand tissues. The ligand may also be tested in a variety of gene deliverymethods, such as ligand-polylysine/DNA complexes (Sosnowski et al., J.Biol. Chem. 272:33647-33653, 1996) or retargeted adenovirus genedelivery (Goldman et al., Cancer Research 57:1447-1451, 1997).

The specificity of the targeting ligand may alternatively be determinedin vivo by biodistribution analysis using one of the reporter genesdescribed herein, such as luciferase. At various time points, miceinjected with the ligand displaying phage are sacrificed and tissuesexamined for the presence of phage in non-targeted tissues byimmunohistochemistry, an enzymatic assay that detects reporter productactivity, or the like.

III. USES

The methods described herein are designed to select cDNAs, Fabs, sFv,random peptides, and the like for discovery of new ligands oranti-ligands. They can also be used to select mutated and gene-shuffledversions of known ligands for targeting ability.

These ligands may have increased transduction efficiency (as measured byan increase in the percentage of infected cells that express thereporter gene); increased expression of the reporter gene (as measuredby intensity of reporter gene expression) in the phage transduced cells;increased specificity of transduction for target cells (as measured forligand specificity); increased stability of the ligand (as measured byability to target the ligand in vivo to tumor cells); increased affinityfor receptor (e.g., removing dimerization requirements for ligands thatdimerize); elimination of the need for cofactors (e.g., development ofan FGF variant that binds with high affinity to the FGF receptor but notto heparin); altered specificity for receptor subtypes (e.g., an FGFvariant that reacts with only one of the four FGF receptors).

The ligands identified by the methods described herein may be used astargeting agents for delivering therapeutic agents to cells or tissues.For example, a therapeutic gene can be incorporated into the phagegenome and delivered to cells via phage bearing the gene delivery ligandon its protein coat.

A transducing gene, as used herein, refers to a gene which encodes adetectable product in the target cell. Preferentially, the transducinggene is a therapeutic gene. A “therapeutic nucleic acid” or “therapeuticgene” describes any nucleic acid molecule used in the context of theinvention that effects a treatment, generally by modifying genetranscription or translation. It includes, but is not limited to, thefollowing types of nucleic acids: nucleic acids encoding a protein,ribozyme, antisense nucleic acid, DNA intended to form triplexmolecules, protein binding nucleic acids, and small nucleotidemolecules. As such, the product of the therapeutic gene may be DNA orRNA. These gene sequences may be naturally-derived sequences orrecombinantly derived. A therapeutic nucleic acid may be used to effectgenetic therapy by serving as a replacement for a defective gene, byencoding a therapeutic product, such as TNF, or by encoding a cytotoxicmolecule, especially an enzyme, such as saporin. The therapeutic nucleicacid may encode all or a portion of a gene, and may function byrecombining with DNA already present in a cell, thereby replacing adefective portion of a gene. It may also encode a portion of a proteinand exert its effect by virtue of co-suppression of a gene product.

As discussed above, the therapeutic gene is provided in operativelinkage with a selected promoter, and optionally in operative linkagewith other elements that participate in transcription, translation,localization, stability and the like.

The therapeutic nucleotide composition of the present invention is fromabout 20 base pairs to about 100,000 base pairs in length. Preferablythe nucleic acid molecule is from about 50 base pairs to about 50,000base pairs in length. More preferably the nucleic acid molecule is fromabout 50 base pairs to about 10,000 base pairs in length. Even morepreferably, it is a nucleic acid molecule from about 50 pairs to about4,000 base pairs in length.

The ligands/anti-ligands provided herein are useful in the treatment andprevention of various diseases, syndromes, and hyperproliferativedisorders, such as restenosis, other smooth muscle cell diseases,tumors, such as melanomas, ovarian cancers, neuroblastomas, pterygii,secondary lens clouding, and the like. As used herein, “treatment” meansany manner in which the symptoms of a condition, disorder or disease areameliorated or otherwise beneficially altered. Treatment alsoencompasses any pharmaceutical use of the compositions herein. As usedherein, “amelioration” of the symptoms of a particular disorder refersto any lessening, whether permanent or temporary, lasting or transient,that can be attributed to or associated with administration of thecomposition.

In certain embodiments, the compositions of the present invention may beused to treat angiogenesis-dependent diseases. In these diseases,vascular growth is excessive or allows unwanted growth of other tissuesby providing blood supply. These diseases include angiofibroma,arteriovenous malformations, arthritis, atherosclerotic plaques, comealgraft neovascularization, delayed wound healing, diabetic retinopathy,granulations due to bums, hemangiomas, hemophilic joints, hypertrophicscars, neovascular glaucoma, nonunion fractures, Osler-weber syndrome,psoriasis, pyogenic granuloma, retrolental fibroplasia, scleroderma,solid tumors, trachoma, and vascular adhesions.

By inhibiting vessel formation (angiogenesis), unwanted growth may beslowed or halted, thus ameliorating the disease. In a normal vessel, asingle layer of endothelial cells lines the lumen, and growth of thevessel requires proliferation of endothelial cells and smooth musclecells.

As well, the ligands, anti-ligands, and cells identified by the presentinvention may be used to treat tumors. In these diseases, cell growth isexcessive or uncontrolled. Tumors suitable for treatment within thecontext of this invention include, but are not limited to, breasttumors, gliomas, melanomas, prostate cancer, hepatomas, sarcomas,lymphomas, leukemias, ovarian tumors, thymomas, nephromas, pancreaticcancer, colon cancer, head and neck cancer, stomach cancer, lung cancer,mesotheliomas, myeloma, neuroblastoma, retinoblastoma, cervical cancer,uterine cancer, and squamous cell carcinoma of skin. For suchtreatments, ligands are chosen to bind to cell surface receptors thatare generally preferentially expressed in tumors.

Through delivery of the compositions of the present invention, unwantedgrowth of cells may be slowed or halted, thus ameliorating the disease.The methods utilized herein specifically target and kill or haltproliferation of tumor cells having receptors for the ligand on theirsurfaces.

The identified ligands/anti-ligands may also be used to treat or preventatherosclerosis and stenosis, a process and the resulting condition thatoccurs following angioplasty in which the arteries become reclogged.Generally, treatment of atherosclerosis involves widening a stenoticvascular lumen, permitting greater blood flow and oxygenation to thedistal tissue. Unfortunately, these procedures induce a normal woundhealing response in the vasculature that results in restenosis. Of thethree components to the normal vascular response to injury, thrombosis,elastic recoil and smooth muscle cell proliferation,anti-thrombotics/platelet inhibitors and vascular stents effectivelyaddress acute/subacute thrombosis and elastic recoil, respectively.However, no existing therapy can modify the vascular remodeling that isdue to proliferation of smooth muscle cells at the lesion, theirdeposition of extracellular matrix and the subsequent formation of aneointima. Accordingly, phage could be used to deliver therapeuticnucleic acids or proteins that would inhibit restenosis.

Wound response also occurs after other interventions, such as balloonangioplasty of coronary and peripheral vessels, with or withoutstenting; carotid endarterectomies; vein grafts; and synthetic grafts inperipheral arteries and arteriovenous shunts. Although the time courseof the wound response is not well defined, if the response can besuppressed for a short term (approximately 2 weeks), a long term benefitis achieved.

The present invention provides the capability of identifying ligandswhich internalize as well as proteins, antibodies, cell/cell interactingproteins that define the interrelationships between cells,host/pathogen, tumor/stroma, autocrine/paracrine factors and allowsidentification of molecules that are targets for new drug discovery orare themselves therapeutically or diagnostically useful.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Modified Phage Vectors for Mammalian CellTransduction

A mammalian expression cassette is inserted into a phage or phagemidvector and is used to detect ligand mediated phage entry via reportergene expression in mammalian cells. A type 3 filamentous phage vector ismodified for transduction of mammalian cells by insertion of a GFPexpression cassette consisting of a CMV mammalian transcriptionalpromoter, the green fluorescent protein gene from pEGFP-N1 (Clonetech;Palo Alto, Calif.), and a bovine growth hormone transcriptionalterminator and polyadenylation signal to make the vector, MEGFP3 (seeFIG. 1A). The mammalian expression cassette also contains an SV40 originof replication adjacent to the CMV promoter. Similar constructs formonitoring entry and subsequent expression of phage genomes in mammaliancells are constructed from other known phage or phagemid vectorsincluding pCANTAB 5 E (Pharmacia Biotech; Piscataway, N.J.) or M13 type3 or 33 for gene III fusions (see Kay et al., Phage Display of Peptidesand Proteins: A Laboratory Manual, Academic Press, 1996; McConnell etal., Mol. Divers. 1:165-176,1996) and M13 type 8 or 88 vector forfusions to gene VIII protein (Roberts et al., Methods Enzymol.267:68-82, 1996; Markland et al., Gene 109:13-19, 1991).

Example 2 Construction of FGF2-Containing Phage Display Vectors

In the following examples, a phage that displays FGF2 on its surface isused to bind to the FGF2 receptor on mammalian cells and beinternalized. An FGF2 gene is subcloned into the modified M13 phage type3 vector, MEGFP3, to create the ligand display phage, MF2/1G3 (see FIG.1B). The gene may also be mutated such that it encodes an FGF2 (C96S)(C78S) double mutant which enhances expression efficiency. The MEGFP3vector has been modified with a mammalian expression cassette designedto express the reporter gene GFP to monitor mammalian cell transductionby the phage. Other vectors include pCANTAB 5 E (Pharmacia Biotech;Piscataway, N.J.) or M13 type 3 or 33 for gene III fusions (see Kay etal., Phage Display of Peptides and Proteins: A Laboratory Manual,Academic Press, 1996; McConnell et al., Mol. Divers. 1:165-176,1996).Similarly, FGF2 is cloned into M13 type 8 or 88 vector for fusion togene VIII protein (Roberts et al., Methods Enzymol. 267:68-82, 1996;Markland et al., Gene 109:13-19, 1991).

To facilitate cloning, the FGF2 gene is amplified by PCR usingoligonucleotide primers that contain appropriate restrictionendonuclease sites in the phage vector gene III or VIII genes. Theresulting phage express FGF2 on their surface coat as detected byanti-FGF2 antibodies in Western blots (FIG. 2) and by ELISA (FIG. 3).

Western blot detection of FGF2-pIII fusion utilizes extracts fromequivalent phage titers of purified FGF2 phage and control phage(MEGFP3) separated by polyacrylamide gel electrophoresis and blottedonto nitrocellulose. FGF2 and FGF2-fusion phage are detected with ananti-FGF2 monoclonal antibody (Transduction Labs; Lexington, Ky.) andHRP conjugated anti-mouse secondary antibody (American Qualex; SanClemente, Calif.) with chemiluminescent development. A single proteinband is detected in the cesium chloride purified FGF2-phage extractmigrating at about 80 kDa. This is about the size predicted for theFGF2-pIII fusion protein (FGF2 (18 kDa) fused to pIII (migrates ˜60kDa)). CsCl purification is performed to remove any non-covalently boundFGF2 fusion protein from the phage particles.

Binding of the FGF2 fusion phage to FGF2 receptor is assessed by ELISAin which recombinant FGF2 receptor is attached to the solid phase and ananti-phage antibody is used as the primary detection antibody. Briefly,phage were captured with an anti-FGF2 rabbit polyclonal antiserum boundto the plate well. An HRP conjugated anti-M13 antibody (PharmaciaBiotech; Piscataway, N.J.) was used to detect the bound phage. Whenanti-phage antibody is used to capture the phage and equivalent OD isobserved for both control (MEGFP3) and FGF2-phage (MF2/1G3) indicatingthat equivalent phage particles are applied to the plate (FIG. 3A). InFIG. 3B an increased OD indicates the presence of FGF2 on the MF2/1G3FGF2-phage.

Example 3 Target Cell Line Engineering

To increase the sensitivity of the assay for transduction by liganddisplay phage the target cell line is transfected with a plasmid that isdesigned to express the SV40 large T-antigen (i.e. pSV3neo). Thisplasmid also contains a drug selection gene such as neomycinphosphotransferase (neo) which confers resistance to the antibiotic G418in stabley transfected mammalian cells. Following transfection of thetarget cell line with plasmid DNA using standard methods (i.e. CaPO₄co-precipitation) the cells are split and maintained in G418 containingmedia until drug resistant colonies appear. The colonies are expanded totest for SV40 T-antigen synthesis by western blotting orimmunoprecipitation using a suitable antibody. Examples of T-antigenexpressing target cell lines are: BOS (BHK with SV40 T-Ag) for screeningFGF variants; HOS-116 (HCT 116 with SV40 T-Ag) for screening peptidesthat target human colon carcinoma; AOS-431 (A431 with SV40 T-Ag) forscreening EGF variants (all parent cell lines are available from ATCC,Manassas, Va.)

Example 4 Binding and Internalization of FGF2-Expressing Phage

The FGF2-expressing phage are also assayed for high affinity receptorbinding and internalization in receptor bearing cells byimmunolocalization and fluorescence microscopy (Hart, J. Biol. Chem.269:12468-12474, 1994; Barry et al., Nature Med. 2:299-305, 1996; Li,Nature Biotech. 15:559-563, 1997).

Infection of mammalian cells with FGF2-expressing phage is performedunder conditions that block entry of wild type M13 phage into cellsexcept chloriquine is not used (Barry et al., supra). Phage are addeddirectly to cells at titers of ≦10¹⁰ CFU/ml in PBS with 0.1% BSA orother suitable blocking agents and incubated at 37° C. or on ice for atleast 1 hour. The cells are then washed extensively in PBS, fixed in 2%paraformaldehyde, and permeabilized in 100% methanol at room temperaturefor 10 minutes. Cells are incubated with rabbit anti-M13 antibody(Sigma; St. Louis, Mo.) in PBS/BSA for 1 hour. The primary antibody isdetected with a phycoerythrin labeled anti-rabbit antibody (LifeTechnologies (Gibco BRL); Rockville, Md.). Surface bound (incubated onice) or internalized (37° C. incubation) phage are detected byfluorescence microscopy.

Example 5 Transduction of Mamialian Cells by FGF2-Ligand Display Phage

FGF2 display phage (MF2/1G3) and an identical phage that lacks the FGF2gene (MEGFP3) are compared for receptor mediated internalization andreporter gene expression in COS cells. The phage are incubated with thecells for 4 hours at 37° C. in DME (Dulbecco's modified Eagles medium,Life Technologies (Gibco BRL); Rockville, Md.) containing 2% BSA (bovineserum albumin) as a blocking agent. After washing to remove unboundphage the cells are returned to the incubator for an additional 72hours. Transduction is measured by counting GFP positive autofluorescentcells. As shown in FIG. 4B, the FGF2 display phage result in about a 10fold greater transduction efficiency than the control phage indicatingthat the displayed FGF2 ligand on the surface of the phage particlesresults in receptor mediated binding and internalization of phage withsubsequent expression of the phage reporter gene. The specificity of theFGF2-phage mediated transduction is demonstrated by successfulinhibition of transduction with excess free FGF2 (2 μg/ml) (FIG. 4B).The low level nonspecific uptake and transduction by the control phage(MEGFP3) is not affected by the presence of excess FGF2.

It is important to show that the MEGFP3 control phage is equally capableof transducing mammalian cells as the display phage when appropriatelytargeted. To compare the transduction ability of both the FGF2-phage andthe control phage, equivalent titers of each phage were used totransfect COS cells using a avidin-biotin FGF2 targeting method. In thismethod biotinylated FGF2 is contacted with the cells and used to capturephage particles via the addition of avidin and a biotinylated anti-phageantibody. The phage/FGF2/ cell binding is performed on ice, unboundphage removed by washing, cells returned to the incubator at 37° C., andtransduction assessed at 72 hours. As seen in FIG. 4A, there is nosignificant difference in transduction between FGF2-phage and controlphage when FGF2 is attached to the phage via an avidin biotin linkage.In this case the biotinylated FGF2 is in excess of the FGF2 displayed onthe phage surface such that internalization is expected to be primarilyvia the biotinylated FGF2. These data demonstrate specific receptormediated transduction of mammalian cells by filamentous phage thatgenetically display a targeting ligand (FGF2).

Example 6 Construction of a Reporter Gene and a Drug Resistance Gene inPhage Display Vectors

A GFP expression cassette consisting of the GFP gene (Cormack et al.,Gene 173:33-37, 1996) under control of a CMV promoter, a neomycinphosphotransferase gene under control of the SV40 early gene promoter,and an SV40 origin of replication are cloned into a gene III phagemidvector such as pCANTAB 5E using standard methods (Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989).The resulting phage is designated pmaM13. The same phagemid genome alsocontaining FGF2-3 fused to gene III is designated pFGF-maM13. Similarconstructs are also made with M13 phage type3 and type 33 and gene VIIIphagemid and phage vectors. Recombinant phage displaying FGF2 on thecoat and carrying the mammalian expression cassettes including the SV40replication origin are prepared by phagemid rescue with M13K07 (orsuitable helper phage) are added to COS cells as described above. GFPexpression is detected by fluorescence microscopy, fluorometry, and flowcytometry at 48-96 hours after phage addition. Drug resistant cells areselected with G418.

Example 7 Selection of FGF2-Expressing Phage from a Mixed Population

A M13 phage display library of random or unknown sequences is spikedwith pFGF-maM13 phage. The mixture is used to infect COS cells asdescribed above. The cells are washed extensively to removenon-specifically bound phage. Cells are re-plated 48-96 hours later at a1 to 10 dilution and grown in G418 to select only cells that receive thetransducing phage gene. Alternatively, the GFP expressing cells areisolated by flow cytometry using an excitation wavelength of 488 andemission wavelength of 510.

DNA is extracted from G418-resistant cells and the FGF2 sequence isamplified. The amplification primers have sequences complementary tophage sequences located on each side of the FGF2 sequence in the geneIII coding sequence. Detection of the FGF2 sequences in selected COScells that are infected with a mixture of phage where the pFGF-maM13phage is diluted at least 1:10,000 with the random sequence phagelibrary demonstrates feasibility of the technique.

Example 8 Identification of FGF2 Variants for Improved Gene Delivery

A library of shuffled FGF2 mutants is created using the gene shufflingmethod described by Stemmer (supra). The FGF2 gene is amplified by PCRand fragmented by DNAse 1 treatment. The fragments are reassembled usingPCR in the absence of primers. The reassembled gene is cut with theappropriate restriction by enzymes and cloned into an M13 phage vectorsuch that the FGF mutants are fused in-frame with the pIII coat proteingene. The phage vector contains a CMV promoter driven GFP reporter geneand an SV40 origin of replication. Several individual phage clones aresequenced to confirm that an average of 3 mutations per phage have beengenerated during the reassembly process. The resulting phage library ofFGF2 mutations is amplified by standard protocols. The target cell line,BOS (BHK with T-Ag) is incubated with the library such that each memberof the library is at an m.o.i. of at least 10. Accordingly, 10¹¹ phagerepresenting 10⁶ copies of 10⁵ individual phage species are applied to10⁵ cells. The phage are incubated with the cells in PBS supplementedwith 2% fetal bovine serum for 1-3 hours, after which non-binding phageare removed by extensive washing with PBS. Media is added and the cellsreturned to the incubator at 37° C. to allow phage internalization.

Example 9 Screening Libraries for Gene Delivery Ligands

If the source of the desired ligand is not known, random peptidelibraries or a cDNA library from placenta is used as a starting pointfor cDNA library screening. The library is amplified in the maM13-33phage by infecting DH5αF′ (or other suitable host) bacteria, growing theculture overnight at 37° C. and isolating the phage from the culturemedium using standard protocols. A cDNA library containing 10⁵ membershas each member represented 10⁶ times in a typical phage titer of 10¹¹colony forming units/ml. The amount of phage used to infect is adjustedto the complexity of the library.

The completed maM13 phage library is screened against the target tissueor cell line. Screening can be performed in-vitro or in-vivo. Thecriteria for a positive “hit” is that the phage must be able to bind, beinternalized, translocate to the nucleus, uncoat and replicate andexpress the genomic DNA containing the reporter gene in the target cell.Thus, only transduced target cells are selected either by GFP expressionand cell sorting or drug resistance. Screening is performed directlyagainst the target cells with no prescreening or enrichment. Enrichmentfor cell binding is performed if no hits are found in the initialscreen. A prescreen to select out phage that bind non-specific cellssurface proteins is performed to reduce non-specific hits or if thereare too many initial hits. Infection of at least 10⁷ target cells isperformed with at least 10¹¹ phage. The cells are incubated for at least2 hours in PBS and washed extensively as described by Barry (Barry etal., Nature Med., 2:299-305, 1996). The cells are incubated in media at37° C. for 48-96 hours and selected in the appropriate drug (e.g., G418)for 7-14 days or until resistant colonies are visible microscopically.Drug resistant colonies are pooled, and the selected cDNAs amplified andsubcloned back into the maM13-33 phage vector using PCR and standardmolecular biology methods. Alternatively individual colonies arescreened. Representative phage clones are sequenced to identifypotential gene delivery ligands. Repeated rounds of infection andselection are performed to reduce the complexity of the selected clones.More stringent screening conditions such as increased selective drugconcentrations or FACS sorting or the strongest fluorescent cells areperformed in the later screens to select the most highly efficient genedelivery ligands from the initial screening.

Screening in-vivo is performed using methods previously described byPasqualini for targeting organs or xenograft tumors using phagedisplayed peptides (Pasqualini, R. et al., Nature Biotechnology, 15,542-546 (1997); Pasqualini, R. et al., Nature, 380, 364-366 (1996))except that the organs or tumors are examined for reporter geneexpression instead of the presence of phage. The phage library isinjected intravenously into mice and organs or tumor samples tested forreporter gene function at 48-96 hours after injection. Tumor cells arecultured in G418 or FACs sorted (for GFP expression) to enrich for cellsthat express the phage transducing gene. The ligand encoding sequencesare amplified from selected cells using PCR as described for in-vitroscreening. As in in-vitro screening, repeated rounds of infection andrescreening are performed at increasing screening stringency to obtainthe most efficient gene delivery ligands.

Example 10 Identification of Ligands that Target Colon Carcinoma

In this example, a library of oligonucleotides encoding random peptidesis inserted into a filamentous phage genome such that the peptides arefused to the C-terminus of intact pIII coat proteins. A type 3 phagevector that only contains one copy of the pIII gene is used and,therefore, all of the pIII protein that is made will be fused to apeptide. Thus, 3-5 copies of a peptide is displayed on each phage. Tosimplify the screening the complexity of the library is first reduced byscreening it for internalizing peptides. Peptides that facilitate theinternalization of phage into a colon carcinoma cell line are isolatedthrough several rounds of selection. The phage library is incubated withthe cells for 3 hours at room temperature. The cells are washedextensively in PBS. A brief proteinase K treatment is used to inactivatephage that adhere to the cell surface. The cells are then lysed and celllysates incubated with host bacteria. Internalized phage are amplifiedin bacteria and subjected to 4 or more iterations of exposure to cellsand recovery of internalized phage. Replicative form DNA is preparedfrom the resulting sublibrary of internalizing phage. The randomsequences in the sublibrary are subcloned into a phage vector MEGFP2that contains a copy of the CMV driven reporter gene (GFP) and an SV40replication origin. MEGFP2 differs from MEGFP3 (FIG. 1A) in that theori-CMV/EGFP expression cassette is in the reverse order, EGFP isfollowed by an SV40 polyadenylation site instead of Bovine GrowthHormone poly A, and the vector contains three additional Nco I siteswithin the ori-CMV/EGFP expression cassette.

The resulting CMV-GFP modified sublibrary is incubated with the HOS-116recipient cell line such that each member of the library is representedat least 10⁶ times. Thus, for example, a library with 10⁵ members isadded to ˜10⁵ cells at a titres of ˜1×10¹¹ yielding an m.o.i. for eachmember of at least 10. The phage are incubated with the cells in PBSsupplemented with 2% fetal bovine serum for 1-3 hours, after whichnon-binding phage are removed by extensive washing with PBS. Media isadded and the cells returned to the incubator at 37° C. to allow phageinternalization.

Example 11 Recovery of Ligand Encoding Sequences from Replicative Phage

At 72 hours following the addition of the phage library. The targetcells are removed from the plate and sorted for GFP expressing cells byFACS. The positively sorted cells are lysed and treated with proteinaseK. The proteins are extracted with phenol/chloroform (24:1 solution) andnucleic acids precipitated in ethanol. The resulting DNA is resuspendedin S1 nuclease buffer and treated with S1 nuclease to removenon-replicative single strand phage DNA. The DNA is again extracted withphenol/chloroform, precipitated, and resuspended in polymerase chainreaction buffer. Alternatively, nuclei are prepared from the positivecells, proteinase K treated and the lysate used directly in the PCRreaction. In either case, an equivalent number of negatively sortedcells are treated in parallel and used in the PCR reaction to monitorthe enrichment of replicative phage DNA (double-stranded) overnon-replicative phage DNA (single stranded) such that there is no phageDNA amplified in the samples from GFP negative cells. If phage DNA isamplified from negatively sorted cells then conditions must be made morestringent for the removal of single stranded phage DNA such asincreasing treatment with S1 nuclease or further purification of nucleithrough repeated sucrose step gradient purification or other suitablemethods known for purification of nuclei (to remove non-replicativephage). These conditions might need to be determined empirically foreach cell line and library used.

The phage sequence(s) encoding the ligand peptide is amplified using anappropriate set of oligonucleotide primers that flank the ligandencoding DNA sequence inserts that is fused to the pIII gene. Theseamplified inserts are recloned into the parent phage vector to create asub-library of phage enriched now for gene delivery ligands for thetarget colon carcinoma cell line. Sequencing is performed onrepresentative clones to determine the complexity. The screening processis reiterated until the complexity is reduced sufficiently to identifyone or more targeting ligands.

Example 12 Second Generation Screening of Peptides

Peptides are selected which have been previously identified from arandom library by one or more panning or screening procedures usingconventional vectors and panning methods (see Kay et al., Phage Displayof Peptides and Proteins: A Laboratory Manual, Academic Press, 1996).The DNA encoding the selected peptides is inserted as a fusion to thepIII coat protein in the MEGFP2 vector containing the GFP reporter genecassette.

An M13 phage random peptide library is screened for peptides that bindand internalize in an FGF receptor overproducing cell line, Flg37 (anFGFR1 stable transfectant of L6 cells (available from the ATCC;Manassas, Va.) obtained from Dr. Murray Korc, UCI; Irvine, Calif.). Inaddition, such a cell line may be easily created by those skilled in theart. Following 5 rounds of panning and rescreening the complexity of thelibrary is reduced such that 80% of the phage are represented by asingle peptide-pIII fusion. The resulting peptide, FL5, has the sequenceFVPDPYRKSR (SEQ ID NO: 1). The same library is also screened againstFlg37 cells by selecting infective phage particles that internalize andassociate with nuclei and cytoskeletal proteins. The 2 predominantpeptide sequences identified by this screen after 5 rounds of panningare FN5A, CGGGPVAQRC (43%) (SEQ ID NO: 2) and FN5B, CLAHPHGQRC (34%)(SEQ ID NO: 3).

Oligonucleotides encoding the 3 peptides are inserted into the MEGFPvector as fusions to the pIII coat protein. The resulting phage are usedto transfect COS cells. Phage are added to cells and incubated overnightat 37° C. in medium with 10% fetal calf serum. The cells are washed toremove unbound phage and returned to the incubator. Transduction isassessed by counting GFP expressing autofluorescent cells at 72 hoursafter the addition of phage. The results (FIG. 5 are that a greatertransduction efficiency is observed with FL5 than FN5A or FN5Bindicating that FL5 is a more efficient as a gene transfer ligand inthis system. The transduction screening method as a second generationscreen is capable of distinguishing among peptides that were selected bydifferent primary cell based screens.

Example 13 EGF Mediated Mammalian Cell Transduction

Epidermal growth factor displaying phage were constructed as describedabove for FGF displaying phage. Western blot analysis demonstrates thatEGF was efficiently expressed on the phage coat in a multivalent manner(FIG. 6). Phage were prepared for Western analysis by obtaining theEGF-phage from cultures of infected host bacteria and purified by PEGprecipitation and CsCl gradient centrifugation. The phage particleproteins were then separated by gel electrophoresis and blotted onto anitrocellulose membrane. Blots were then probed with either anti-EGF oranti-pIII antibody (mouse anti-human EGF, Biosource International;Camarillo, Calif.) or anti-pIII antibody (mouse anti-pIII, MoBiTech;Germany) followed by HRP-goat-anti-mouse (Jackson Laboratories, USA).

Following the procedures detailed above, EGF-phage were screened fortheir ability to effectively transduce COS cells. Briefly, EGF-phagewere incubated with COS cells (˜75,000 cells/well) for 72 hours with avariety of phage titers. As demonstrated by FIG. 7 the optimal dose was10¹⁰ pfu/ml which resulted in the highest transduction efficiency withalmost no non-specific transduction by untargeted phage. Transductionefficiency also increases with longer incubation times. As demonstratedin FIG. 8 when EGF-phage were incubated with COS cells (˜75,000cells/well) at 10¹¹ pfu/ml for various times and subsequently measuredfor GFP expression at 72 hours, longer incubation times increasedtransduction efficiency.

Further, specificity of EGF-phage mediated COS cell transduction wasdetermined by incubating EGF-phage with excess ligand. As depicted inFIGS. 9A and 9B, COS cells incubated with 10¹¹ pfu/ml of phage for 72hours with or without excess ligand or untargeted phage demonstrate thattargeting is due to the presence of the ligand.

Example 14 Simultaneous Identification of Internalizing Ligands andAnti-Ligand Binding Targets

To identify internalizing ligand-anti-ligand binding targetinteractions, the putative ligand is displayed on the surface offilamentous phage that carry a mammalian reporter gene expressioncassette. The candidate binding target peptides/proteins are expressedon the surface of COS cells by substituting the target cDNA for theextra cellular domain encoding DNA portion of the EGF receptor in asuitable mammalian cell expression vector (i.e., pcDNA 3.1; Invitrogen,Calif.).

To accomplish this, a library of cDNAs is inserted into a mammalianexpression vector (pcDNA 3.1) such that the cDNAs are fused to thetransmembranes and intracellular domains of EFG receptor cDNA. DNA isprepared from individual or pools of bacterial clones that have beentransformed to carry the cDNA-receptor fusion protein expressionplasmid. COS cells are transfected with the resulting plasmid DNAs insix well plates at low density. At 24 hours later, ligand display phagecarrying the CMV driven reporter gene GFP are added to the transfectedCOS cells.

Binding of the phage displayed ligand to the cell surface displaybinding target (i.e. protein—EGF receptor fusion protein), results indimerization of the receptor and subsequent internalization of phagethat display the binding ligand. The internalized phage are traffickedto the nucleus where the reporter gene is expressed. 72 hours afteradding phage, cells expressing the reporter gene are selected by FACs.cDNAs encoding reactive peptides are identified by the presence of GFPpositive cells in the COS transfectants for each cDNA or cDNA pool. Thebinding ligand is identified by PCR amplification and sequencing of thephage ligand-pull fusion gene. The target peptide is identified by PCRamplification and sequencing the peptide-EGF receptor fusion proteinfrom the selected cell(s).

Example 15 Identification of Cell Targets

Phage that display a ligand as a pIII fusion on the phage coat and carrythe GFP expression cassette are prepared using standard protocols, asdiscussed above. Control phage that carry GFP but don't display a ligandare also prepared. Candidate cell targets are seeded into 6 well cultureplates at about 40,000 cells/well. At 24 hours after seeding cells,phage are added at ˜10¹⁰ pfu/ml. The plates are incubated at 37° C. foran additional 72 hours. Each cell well is scored by counting GFPpositive autofluorescent cells. The cell types that have a ratio of GFPpositive cells in the ligand-phage treated well/control phage treatedcells of greater than 1.0 are selected as targets for further study andcharacterization. As an alternative to GFP, a drug resistance gene canbe used in which case after 72 hours the cells are allowed to continuegrowth in selective medium containing the drug. Positive cell types arescored by counting wells that have drug resistant colonies.

Carcinoma cell lines which are known to express EGF were screened by theabove method using EGF-phage and compared to the control endothelialcell line which is EGF receptor negative (Cell lines obtained from ATCC,Manassas, Va. and grown under standard ATCC culture conditions). Asshown in FIG. 10, the carcinoma cell lines derived from various tissueswere differentially transduced while the receptor negative, endothelialcells displayed no transduction. Accordingly, identification of targetcells or tissues can be accomplished using these methods.

Example 16 Identification of Pathogen Target Cells

Ligand display phage are constructed as discussed above, with the ligandbeing full-length or fragments of coat or envelope proteins of a knownor suspected pathogen. The ligand expressed on the display phage coatcan be expressed from the cDNA or cDNA derivative of the coat orenvelope protein of a known or suspected pathogen (e.g., HIV envelopeprotein gene). The envelope gene is randomly fragmented to form alibrary of display phage display distinct portions of the coat protein.Thereby allowing determination of the portion of the gene that encodes aprotein that functionally interacts with the host cell surface receptorsallowing internalization. Smaller pathogen coat proteins are displayedin entirety. The pathogen coat display phage acts as surrogate pathogenwith the advantage of providing a simple assay for detection of hostcells. Phage displaying coat protein are screened against various celltypes in vitro as described above or in vivo by injection and subsequentidentification of target cells and tissues by fluorescent microscopy,FACS analysis to detect GFP, or growth in selective medium to detectexpression of a drug resistance marker.

Example 17 Identification of Pathogen Ligands

The gene(s) or portion of a gene that interacts with the host cellsurface receptor to allow internalization is identified by making aphage display library of the cDNAs expressed by the pathogen or of thepathogen genome or fragments of the genome. The display library phagevector carries the GFP or suitable reporter gene driven by the mammalianpromoter, as described above. The libraries are then screened against aknown or putative host cell types by detecting transgene expression(i.e., drug selection or other detectable marker). Once cells areidentified, the sequence of the nucleic acid encoding the internalizingligand is determined by PCR sequencing of the pIII-putative ligandfusion construct.

Example 18 Identification of Secreted and Internalizing Ligands forTumor Cells

Tumor cells interact with surrounding host stromal and other cell typesvia chemo-attractants and other factors which, for example, stimulatethe stromal cells to secrete factors that support tumor growth (i.e.,VEGF). To investigate these interactions, a library of putative secretedligand cDNAs is prepared tumor cell mRNA and selected by methods knownin the art such as epitope-tagging, Sloan et al., Protein Expression andPurification 11:119-124, 1997. The secreted protein encoding cDNAs areinserted into the reporter phage vector as described above.

Individual phage clones or pools of phage clones are screened againstvarious stromal cell types to identify cell types that are targets fortumor cell secreted factors, and to identify the secreted factors. Theinverse strategy can also be applied by screening a library of, forexample, fibroblast or other stromal cell secreted protein encodingcDNAs for factors that bind and internalize into various tumor celltypes.

Example 19 Selection of EGF-Expressing Phage from a Population ofNon-Targeted Phage

Non-targeted M13 phage was spiked with EGF-phage. The mixture was usedto infect COS cells and incubated for 72 hours, as described above. Thecells are washed extensively to remove non-specifically bound phage. TheGFP expressing cells are isolated by flow cytometry (FACS) using anexcitation wavelength of 488 and emission wavelength of 510.

DNA is extracted from GFP positive cells and the EGF sequence wasamplified by PCR. The amplification primers have sequences complementaryto phage sequences located on each side of the EGF sequence in the geneIII coding sequence. These sequences were re-cloned into the phagevector and new phage were prepared for subsequent rounds of selection.

Briefly, the following biotinylated oligonucleotides were used toamplify the ligand gene III fusion:

Anchor1M8f 48 5′Bio AAAGGATCCGGGTTCCCGCGTGGGCGATGGTTGTTGTCATTGTCGGC (SEQID NO: 5) M3rev2 25 bio CCGTAACACTGAGTTTCGTCACCAG (SEQ ID NO: 6)

However, the following have also been used with success:

M8for2b 30 biotin GCGTGGGCGATGGTTGTTGTCATTGTCGGC (SEQ ID NO: 7) m3revB25 biotin CCACAGACAACCCTCATAGTTAGCG (SEQ ID NO: 8)

Proteinase K treated nuclear preparations of FACsorted COS cells. PCR iscarried out using Clonetech's Advantage GF polymerase mix and cycledunder the following conditions:

1 cycle:

94° C. 1 min.

40 cycles:

94° C. 20 sec.

60° C. 20 sec.

72° C. 20 sec.

Following amplification, duplicate samples were combined and purifiedusing Qiaquick columns (Qiagen Inc., Valencia, Calif.). The PCR productswere then digested with NcoI and PstI restriction endonucleases. WhileSA-magnetic beads or other means for removal of “ends” of fragmentsincreases ligation efficiency, such removal is not required. Thedigestion product is ligated into a new phage vector and used totransform competent cells by electroporation.

After sub-cloning and electroporation of bacterial cells, the bacteriawere plated in top agar and grown overnight at 37° C., plaques wereselected from plates and analyzed via PCR using oligonucleotides asfollows:

MANPgIIIf 20 TTTTGGAGATTTTCAACGTG (SEQ ID NO: 9) MANPgIIIr 20TGCTAAACAACTTTCAACAG (SEQ ID NO: 10)

However, the oligonucleotides listed previously above, are equallyuseful.

As demonstrated in FIG. 11, enrichment of targeted EGF-phage from 0.1%to 100% EGF-phage was complete after 3 rounds of selection andenrichment from 0.0001% to 100% EGF-phage was complete after 4 rounds ofselection. Accordingly, this experiment demonstrates the ability toselect a specific ligand expressing phage from a population at dilutionsof 1:10³ and 1:10⁶. In addition, while further diluted ligand expressingphage can be detected, further rounds of selection may be necessary.

Example 20 Creation of a Sub-Library of Peptides that are Internalizedand Trafficked to the Nucleus

Phage that display a candidate ligand as a pIII or pVIII fusion on thephage coat are prepared using standard protocols, as discussed above. Inthe present experiment a phage library (MANP-TN10, Dyax, Corp.) is used.COS cells are plated on 2×10 cm plates at about 105,000 cells/plate. At24 hours after seeding cells, phage are added at ˜10¹⁰ pfu/ml. Theplates are incubated at 37° C. for an additional 72 hours. The cells arethen harvested in Trypsin-EDTA and pelleted. The cells are re-suspendedin 0.5 ml of PBS and under-layed with 0.5 mls of nuclear isolationbuffer (NIB) and spun at 200× g for 8 minutes at 4° C. (NIB=40% Sucrose,0.1% DMSO, 2% NP40, 1.6% Triton X-100, 0.2 mM AEBSF(4-(2-Aminoethyl)benzenesulfonyl Fluoride, in PBS). The pellet is thenre-suspended in 400 μl 1% NP40 and under-layed with 40% sucrose andagain spun at 200× g for 8 minutes at 4° C.

The pellet is then re-suspended in 100 μl PKB (Proteinase K Buffer—50 mMTris-HCl pH 8.5, 1 mM EDTA, 0.5% Tween-20). 1.4 μl of PK (Proteinase Kfrom Boehringer Mannheim, 14 mg/ml) is added and incubated at 55° C. for3 hours, then heated to 95° C. for 15 min. The DNA is then pelleted atmaximum speed in a microfuge and washed 1 time with 70% ethanol followedby air drying in a hood. The resulting pellet is resuspended in a 20 μlof 10 mM Tris pH 7.2 and the ligand gene III fusion is amplified asdescribed in Example 19, above, except that the cycling program wasadjusted as follows:

1 cycle:

94° C. 1 min.

25 cycles:

94° C. 20 sec.

60° C. 20 sec.

72° C. 20 sec.

Following amplification, the product was purified using a Qiaquickcolumn (Qiagen) followed by digestion using NcoI and PstI restrictionenzymes, as described above. Biotinylated fragments are removed usingstreptavidin conjugated beads (Promega Corp., Madison, Wis.), and theresulting sample is ethanol precipitated. The precipitated DNA is thenwashed and re-suspended in 10 μl of 10 mM Tris pH 8.5 and ligated intothe reporter gene carrying phage vector. Following ligation the DNA isethanol precipitated, washed, and used to transform competent cells(Stratagene electrocompetent cells X11Blue MRF′). Phage selected for inthis manner (TN10 nuclear selection) are then compared to non-selectedphage (TN10 library). The comparison of the pre-selected pooldemonstrates that a significant population of the library which did notinternalize has been removed in one round of screening, see Table below:

Phage Dilution Titer Vol of Phage Fraction Prep In/Out factor μl platedplaques (pfu/ml) prep (μl) recovered of input TN10 In 8 100 123 1.23E +08 5000 6.15E + 11 library TN10 Out 0 10 127 1.27E + 01  400 5.08E + 038.26E − 09 nuclear

Example 21 Ligand Selection Via Nucleic Acid Binding Domains

A phage library comprising a number of ligand displaying phage arecreated using a lac operon containing phagemid. The phagemid can beeither a reporter gene containing phage (e.g., MEGFP3) or a non-reportergene containing phagemid (e.g., any vector containing a phage origin ofreplication, such as pCOMB3, pBS+, pCR, and the like). The MEGFP3 vectorhas been modified with a mammalian expression cassette designed toexpress the reporter gene GFP to monitor mammalian cell transduction bythe phage. Other vectors include pCANTAB 5 E (Pharmacia Biotech;Piscataway, N.J.) or M13 type 3 or 33 for gene III fusions (see Kay etal., Phage Display of Peptides and Proteins: A Laboratory Manual,Academic Press, 1996; McConnell et al., Mol. Divers. 1:165-176,1996).Similarly, the ligand library is cloned into M13 type 8 or 88 vector forfusion to the gene VIII protein (Roberts et al., Methods Enzymol.267:68-82, 1996; Markland et al., Gene 109:13-19, 1991).

Candidate cell targets are seeded into 6-well culture plates at about40,000 cells/well. At 24 hours after seeding cells, phage are added at˜10¹⁰ pfu/ml. The plates are incubated at 37° C. for an additional 72hours. Following incubation with the phage library, the target cells areremoved from the plate and sorted for GFP expressing cells by FACS ordirectly lysed and the nucleic acid purified by passing over a sepharose4B DNA affinity-column having conjugated thereto the lac repressorprotein. Prior to affinity purification the cells are lysed and anuclear extract is produced by following standard procedures, such asthose described by Cull et al., Proc. Nat'l. Acad. Sci. USA89:1865-1869, 1992; Schatz et al., Methods in Enzymology 267:171-191,1996). The nuclear extract is then passed over the affinity column.

After applying to the column, the column is washed extensively withloading buffer (20 mM Tris-HCl pH 7.2) and eluted with a salt gradient.The resulting DNA containing fractions are pooled, amplified by PCRusing the flanking gene III or gene VIII fusion sequences as primertemplates, and subcloned back into a phagemid vector for further roundsof enrichment or alternatively for direct sequence characterization.

Example 23 Internalized Ligand Sequence Amplification by SV40 ShuttleVector Transduction

The phagemid-shuttle vector includes the SV40 origin and packagingsequences, as well as SV40 capsid-encoding late genes under control of apromoter functional in target cells. This vector is created by PCRamplification of relevant sequences or direct restriction enzymedigestion and sub-cloning. These sequences can be obtained fromcommercially available vectors or wild-type virus. Similarly, the phagecoat protein fusion, the phage origin and packaging sequences, andbacterial selection markers can be assembled from current phage vectors.

Phage particles expressing a library of ligands as genetic fusions onthe coat proteins (gIII or gVIII) are generated by rescuing phagemidcontaining bacteria with a helper phage, such that the phagemid genomeis packaged into the phage particle expressing the ligand which isencoded by that genome. A target cell line containing the SV40 Tantigens (either transfected or provided in trans with the VP22 fusionprotein as a delivery vehicle) is incubated with the phage particles.Those particles expressing the appropriate ligands deliver the phagemidDNA to the nucleus. Due to the presence of the large T antigen in thecells and the SV40 origin, the DNA replicates. The dsDNA is thenpackaged into SV40 viral particles due to the presence of the capsidproteins encoded by the SV40 genes also carried on the phagemid genome.The SV40 particles carrying the phagemid then infect other neighboringcells and thus amplifying the internalized ligands until the wholepopulation of cells is infected. Eventually, all cells are lysed due toviral production. Viral particles are harvested from the supernatantsand the DNA they contain is analyzed by sequencing to determine thesequence of the ligand responsible for the initial internalization.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

10 1 10 PRT Unknown Description of Unknown Organism A screened peptide,from a random peptide library, that binds and internalizes in a FGFreceptor overproducing cell line 1 Phe Val Pro Asp Pro Tyr Arg Lys SerArg 1 5 10 2 10 PRT Unknown Description of Unknown Organism A screenedpeptide, from a random peptide library, that binds and internalizes in aFGF receptor overproducing cell line 2 Cys Gly Gly Gly Pro Val Ala GlnArg Cys 1 5 10 3 10 PRT Unknown Description of Unknown Organism Ascreened peptide, from a random peptide library, that binds andinternalizes in a FGF receptor overproducing cell line 3 Cys Leu Ala HisPro His Gly Gln Arg Cys 1 5 10 4 4 PRT Unknown Consensus nuclearlocalization sequence 4 Lys Xaa Xaa Xaa 5 47 DNA Artificial Sequence PCRPrimer 5 aaaggatccg ggttcccgcg tgggcgatgg ttgttgtcat tgtcggc 47 6 25 DNAArtificial Sequence PCR Primer 6 ccgtaacact gagtttcgtc accag 25 7 30 DNAArtificial Sequence PCR Primer 7 gcgtgggcga tggttgttgt cattgtcggc 30 825 DNA Artificial Sequence PCR Primer 8 ccacagacaa ccctcatagt tagcg 25 920 DNA Artificial Sequence PCR Primer 9 ttttggagat tttcaacgtg 20 10 20DNA Artificial Sequence PCR Primer 10 tgctaaacaa ctttcaacag 20

We claim:
 1. A method of selecting internalizing ligands displayed on agenetic package from a library of ligand displaying genetic packages,comprising: (a) contacting a library of ligand displaying geneticpackages with a cell(s), wherein each package carries a gene encoding adetectable product which is expressed upon internalization of thepackage, and (b) detecting product expressed by the cell(s); and therebyselecting internalizing ligands displayed on agenetic package.
 2. Themethod of claim 1, further comprising isolating the cell(s) that expressthe detectable product.
 3. The method of claim 2, wherein the cell(s)are isolated by flow cytometry.
 4. The method of claim 2, furthercomprising recovering a nucleic acid molecule encoding a ligand fromcell(s) expressing the product.
 5. The method of claim 1, wherein thelibrary is a cDNA library.
 6. The method claim 1, wherein the detectableproduct is selected from the group consisting of green fluorescentprotein, β-galactosidase, secreted alkaline phosphatase, chloramphenicolacetyltransferase, luciferase, human growth hormone and neomycinphosphotransferase.
 7. The method of claim 1, wherein the liganddisplaying genetic packages comprise bacteriophage.
 8. The method ofclaim 6, wherein the bacteriophage are filamentous phage.
 9. The methodof claims 7 or 8, wherein the bacteriophage carries a genome vector. 10.The method of claim 1, wherein said method is a high throughput methodand said cells are immobilized on an array.